Effective vaccines potentiate antibody avidity and increase T cell longevity. A new generation of mucosal vaccines is being developed using probiotic Lactobacillus (L.) strains expressing anthrax-protective antigen (PA) fusion protein. Such an oral vaccine, expressed by probiotic Lactobacillus species not only optimally activates dendritic cells (DCs), but also delivers targeted antigens (i.e. anthrax PA) to mucosal DCs via 12-mer peptides derived from a bacteriophage library. We further optimized the expression of the immunogenic vaccine fusions expressed by L. gasseri. Obtained data clearly show that the use of a high copy vector for PA-DC fusion expression by L. gasseri confers robust immune protection against anthrax Sterne challenge. The objective of this proposal is to test the efficacy of such an oral multivalent vaccine consisting of anthrax PA and Clostridium botulinum neurotoxin serotype A (BoNT/A) heavy chain (Hc) in inhaled anthrax and BoNT/A intoxication and lastly to examine whether integration of the anthrax PA-DCpep and/or BoNT/A C-terminus of Hc-DCpep fusions into the L. gasseri chromosome is superior to plasmid-based approaches. Three novel approaches adapted for this work are: (a) improving adjuvanticity of the delivery vector by employing L. gasseri; (b) improving vaccine potency by specifically using a high copy vector for immunogenic fusions; and (c) expressing the multivalent immunogenic fusion vaccines in L. gasseri by bacterial chromosomal insertion and avoiding potential plasmid instability. Our hypothesis is that this optimized oral multivalent vaccine will induce mucosal and systemic immune responses against inhaled anthrax and BoNT/A in mice. The specific aims are: (1) to determine whether L. gasseri expressing targeted anthrax PA-DCpep or BoNT/A Hc-DCpep fusion vaccines enhances protective immunity against inhaled anthrax or BoNT/A intoxication; (2) to determine the protective efficacy of PA-DCpep or BoNT/A Hc-DCpep fusion vaccines in vivo, when expressed from a chromosomal location in L. gasseri; and (3) to determine the efficacy of the probiotic multivalent vaccine consisting of anthrax-PA and BoNT/A-Hc fusions against inhalational anthrax and BoNT/A challenge. This effort will be accomplished via novel adjuvants that provide targeting of the vaccine, evaluation of vaccine effectiveness against deadly pathogens, and the control of probiotic L. gasseri gene expression that can readily be orally consumed to enable natural delivery of targeted antigen to mucosal DCs. Activation of a group of cells in our immune system, called dendritic cells, is critical for proper defense against different types of infection. The goal of this grant proposal is to provide tools for efficient activation of mucosal and systemic immune system using a new generation of harmless immunostimulatory bacteria called Lactobacillus gasseri that can be ingested as dietary supplements. Our data show that L. gasseri producing anthrax vaccine optimally generated robust protective immunity against anthrax infection. Therefore, this probiotic vaccine strategy will be used to show that they are important for multivalent vaccine delivery as well as critical vehicles that stimulate mucosal and systemic immunity for combating deadly bacteria such as anthrax and botulinum.